WO2017147649A1

WO2017147649A1 - Methods of treatment

Info

Publication number: WO2017147649A1
Application number: PCT/AU2017/050173
Authority: WO
Inventors: Peter Hansen; Julien FREITAG; Kiran Shah
Original assignee: Magellan Stem Cells Pty Ltd
Priority date: 2016-02-29
Filing date: 2017-02-28
Publication date: 2017-09-08
Also published as: AU2017225902B2; AU2023201505A1; AU2017225902A1

Abstract

The present invention relates to method of treating a human subject suffering from joint degeneration. The method is based on local administration of human mesenchymal stem cells (e.g., autologous human mesenchymal stem cells) according to a specific therapeutically effective administration regimen that minimizes moderate and severe adverse events. The invention also relates to a method of treating a human subject suffering from joint degeneration, where the method includes performing abrasion osteoplasty on an affected joint followed by local administration to the affected joint of a therapeutically effective number of human mesenchymal stem cells.

Description

METHODS OF TREATMENT

FIELD OF THE INVENTION

The specification relates generally to the field of regenerative medicine. More particularly, the specification relates to methods for treating joint degeneration using human mesenchymal stem cell therapies.

BACKGROUND OF THE INVENTION

The management of conditions associated with joint inflammation and damage, particularly in osteoarthritis, presents a challenge to clinicians. The capacity of joint cartilage to repair, particularly after skeletal maturity, is limited. Cartilage regeneration has an inherently low healing potential due to the avascular nature of cartilage and hence lack of systemic regulation.

There are currently no disease-modifying pharmaceutical therapies for such conditions. Indeed, all currently accepted treatments are aimed primarily at symptom control. Current conservative management strategies fail to alter disease progression and surgical management in the form of joint replacement is associated with significant complications. Alarmingly, there are an increasing number of total joint replacements being performed on patients under the age of 65.

Methods for the repair of articular cartilage lesions, including surgical microfracture and cellular scaffold transplantation, have been investigated with some success in both preclinical and clinical trials. Unfortunately, these techniques are limited to the repair of focal lesions only, and are not easily transferable to joint conditions where there is more generalized loss of cartilage volume. Thus, there is an ongoing need for improved methods for the treatment of joint inflammation and damage.

SUMMARY OF THE INVENTION

The present invention relates to a method of treating a human subject suffering from joint degeneration, which method is based on local administration of human mesenchymal stem cells (hMSCs) to an affected joint according to a specific therapeutically effective dosing regimen that minimises moderate and severe adverse events. Surprisingly, it was found that intra-articular administration of hMSCs, even autologous hMSCs, above a certain number or frequency induces moderate or severe adverse events in a majority of patients notwithstanding the fact that hMSCs are generally considered immuno-neutral.

Accordingly, in a first aspect the present invention provides a method of treating a human subject suffering from joint degeneration, the method comprising local administration to an affected joint of a therapeutically effective number of hMSCs, wherein the number of hMSCs is insufficient to induce a moderate or severe adverse event in the human subject.

The invention also relates to the finding that abrasion osteoplasty of an affected joint prior to local administration of hMSCs results in an enhanced therapeutic effect relative to MSC administration without prior abrasion osteoplasty. Thus, in another aspect the present invention provides a method of treating a human subject suffering from joint degeneration, the method comprising performing:

(i) abrasion osteoplasty on an affected joint; and

(ii) local administration of a therapeutically effective number of human mesenchymal stem cells to the affected joint following abrasion osteoplasty. In an embodiment of this method, the number of hMSCs is insufficient to induce a severe adverse event.

In some embodiments the number of hMSCs is insufficient to induce a severe adverse event or a moderate adverse event in greater than 25% of human subjects treated. In other embodiments the number of hMSCs is insufficient to induce a severe adverse event or a moderate adverse event in greater than 10% of human subjects treated.

In some embodiments of the first aspect, the hMSCs are locally administered to an affected joint that previously underwent abrasion osteosplasty. In other embodiments of the first aspect, the method includes the step of performing abrasion osteoplasty on the affected joint (other than an intervertebral disc) prior to local administration of hMSCs.

In some embodiments the human subject is suffering from a condition selected from, but not limited to, osteoarthritis, rheumatoid arthritis, bursitis, a cartilage tear, intervertebral disc inflammation, or intervertebral disc degeneration. In some embodiments the subject is suffering from osteoarthritis.

In some embodiments the affected joint to which hMSCs are administered is an intervertebral disc. In other embodiments the affected joint is a large joint. In some embodiments the affected large joint is a knee joint. In some embodiments, where the affected joint is a large joint, the therapeutically effective number of mesenchymal stem cells to be administered is about 30 million to about 70 million. In other embodiments the therapeutically effective number of mesenchymal stem cells is about 40 million to about 60 million. In further embodiments the therapeutically effective number of mesenchymal stem cells is about 50 million.

In some embodiments the affected joint is a medium joint or small joint. In some embodiments the medium joint or small joint is selected from, but not limited to, a hip joint, a shoulder joint, an elbow joint, an ankle joint, a Sacro-iliac joint, a wrist joint, a hand joint, a foot joint, a facet joint, or a temporomandibular joint. In some embodiments, where the affected joint is a medium joint or small joint, the therapeutically effective number of hMSCs to be administered is about 5 million to about 20 million. In other embodiments the therapeutically effective number of hMSCs is about 7 million to about 15 million. In further embodiments the therapeutically effective number of hMSCs is about 9 million to about 12 million. In some embodiments the therapeutically effective number of hMSCs is about 10 million.

In relation to any of the above aspects, in some embodiments the hMSCs to be administered are adipose tissue-derived hMSCs or culture-expanded hMSC progeny thereof. In other embodiments the hMSCs are hMSCs derived from bone marrow, dental pulp, skin or culture-expanded hMSC progeny of any of the foregoing.

In some embodiments the hMSCs are autologous human MSCs. In other embodiments the hMSCs are allogeneic hMSCs.

In some embodiments any one of the above-mentioned methods of treatment further includes at least one additional local administration of a therapeutically effective number of hMSCs following the first local administration, but no more than four additional local administrations within 18 months of the first local administration. In some embodiments the interval between the first local administration and a second local administration is about 4 months to about 8 months. In other embodiments the interval between the first local administration and the second local administration is about 6 months. In yet other embodiments rather than providing the at least one additional local administration at a pre-determined fixed interval, the additional local administration is provided following a determination that the subject's joint degeneration is worsening.

In some embodiments any one of the above-mentioned methods of treatment further includes local administration to the joint of a growth factor, cytokine, or glycosaminoglycan. In some embodiments the growth factor or cytokine is selected from, but not limited to, TGF-βΙ, basic fibroblast growth factor (bFGF) and interleukin- 1 receptor antagonist (IL-1RA), or any combination thereof. In some embodiments the glycosaminoglycan is hyaluronic acid (HA). In some embodiments the local administration to the joint of a growth factor, cytokine, or glycosaminoglycan includes local administration of autologous conditioned serum (ACS).

In some embodiments the human subject to be treated underwent microfracture or microdriUing at the affected joint prior to local administration of hMSCs to the affected joint. In some embodiments the method includes the step of performing microfracture or microdriUing on the affected joint prior to the local administration of hMSCs. In some embodiments the affected joint has not undergone microfracture or microdriUing on the affected joint prior to local administration of hMSCs.

In some embodiments the hMSCs to be administered have not been cryopreserved prior to the local administration. In other embodiments the hMSCs to be administered were cryopreserved and thawed prior to the local administration. In some embodiments the hMSCs to be administered are culture-expanded hMSCs, which are obtained from culture expansion of either a cryopreserved seed stock of hMSCs, or from primary hMSCs that were not previously cryopreserved.

In some embodiments the treatment results in one or more of: regrowth of cartilage at the affected joint; a reduction of subject-reported pain in the affected joint of at least about 10% within 12 months of the first administration; and at least about a 10% increase in mobility of the affected joint within 12 months of the administration.

In some embodiments the human subject to be treated is not older than about

60 years. In some embodiments the human subject is about 40 to about 60 years old. In other embodiments the human subject is about 18 to about 50.

In a related aspect the invention provides hMSCs when used for the treatment of joint degeneration in a human subject according to any of the above treatment methods.

In another related aspect the invention provides hMSCs in a dose therapeutically effective for the treatment of joint degeneration in a human by local administration to an affected joint, wherein the dose includes an insufficient number of hMSCs to induce a moderate or severe adverse event.

In a further related aspect the invention provides the use of hMSCs for the manufacture of a medicament for the treatment of joint degeneration in a human, the medicament comprising a therapeutically effective number of mesenchymal stem cells for local administration to an affected joint, wherein the therapeutically effective number of human mesenchymal stem cells is insufficient to induce a moderate or severe adverse event. In another aspect the invention provides hMSCs in a dose therapeutically effective for the treatment of joint degeneration in a human subject by administration of the hMSCs to an affected joint following abrasion osteoplasty.

In a further aspect the invention provides the use of hMSCs for the manufacture of a medicament for the treatment of joint degeneration, wherein the medicament is administered to an affected joint following abrasion osteoplasty.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 - Knee osteoarthritis Monash study patients exhibit reduced knee pain up to 12 months following intra-articular injection of autologous MSCs. Line graphs illustrating numerical pain rating scale (NPRS) scores of pain improvement in all treatment groups over 12 months. A statistically significant improvement in pain was observed in those aged 41-60 years at 1, 3 and 9 months (two tailed t-test; p < 0.05).

Figure 2 - Knee osteoarthritis Monash study patients exhibit improved Western Ontario and McMaster University Osteoarthritis Index (WOMAC) outcomes following intra-articular injection of autologous MSCs. Line graphs illustrating WOMAC outcomes indicating a trend of improvement in pain and function across all treatment groups. A reduced WOMAC score (increased pain and reduced function) was observed in the control group. Using a two-tailed test, statistically significant improvement was noted at 1 and 3 months of follow-up. Figure 3 - Knee osteoarthritis Monash study patients exhibit improved Knee Injury and Osteoarthritis Outcome Score (KOOS) outcomes following a single intra-articular injection of 100 million autologous MSCs. Line graphs illustrating KOOS outcomes of those patients undergoing a single injection protocol of 100 million hMSCs. Analysis of data indicated a trend that patients reported improvements in overall function over 12 months.

Figure 4 - Knee osteoarthritis Monash study patients exhibit improved KOOS assessment following two intra-articular injection of 100 million autologous MSCs each. Line graphs illustrating KOOS outcomes of clinical study patients undergoing a two injection protocol of (100 million hMSCs per injection). A trend is indicated that patients receiving two injections reported greater improvements in overall function over 12 months than patients receiving a single injection.

Figure 5 - Private practice patients with knee osteoarthritis exhibit reduced knee pain following intra-articular injection (2 x 100 million) or (5 x 40 million) of autologous MSCs. Line graphs illustrating NPRS score improvement in all treatment groups over 12 months indicate comparable improvements in both two injection (100 million hMSCs) and five injection (40 million hMSCs/injection) protocols in consented, private practice patients treated outside of the clinical study (Figs. 1-4), but under the same treatment protocol.

Figure 6 - Private practice patients with knee osteoarthritis exhibit improved WOMAC outcomes following intra-articular injection (2 x 100 million) or (5 x 40 million) of autologous MSCs. Line graphs illustrating WOMAC outcomes in all treatment groups over 12 months indicate comparable improvements in both 2 injection and 5 injection protocols in consented, private practice patients treated outside of the clinical study (Figs. 1-4), but under the same treatment protocol.

Figure 7 - Private practice patients with knee osteoarthritis exhibit improved KOOS outcomes following intra-articular injection (2 x 100 million). Line graphs illustrating KOOS outcomes over 12 months indicate improvement in private practice patients receiving a 2 x 100 million hMSC injection protocol. Figure 8 - Private practice patients with knee osteoarthritis exhibit improved KOOS outcomes following intra-articular injection (5 x 40 million). Line graphs illustrating KOOS outcomes over 12 months indicate improvement in private practice patients receiving the 5 x 40 million hMSC injection protocol. The KOOS outcomes in private patients receiving the 5 x 40 million hMSC protocol was not significantly different from those observed in patients receiving 2 x 100 million hMSCs. However, the former had observable increased pain and swelling following each injection and was discontinued as it did not provide greater improvement than the 2 x 100 million hMSC protocol.

Figure 9 - Adverse events observed in knee osteoarthritis patients (Monash study plus private practice) receiving injections of 100 million autologous hMSCs Bar graphs indicating the percentage of mild, moderate, and serious adverse events in clinical study and private practice patients. The bar graph shows that 75% of patients administered 100 million hMSCs reported a moderate or severe adverse event. Among patients experiencing a severe adverse event, two patients needed to be hospitalized for pain relief.

Figure 10 - A reduced number of adverse events is observed in knee osteoarthritis patients (Monash study plus private practice) receiving injections of 50 million (2 x 50 million) autologous hMSCs. Bar graphs indicating the percentage of mild, moderate, and serious adverse events. The graph shows that only 8% of patients receiving two injections of 50 million autologous hMSCs experienced a moderate adverse event with 92% of patients recording either a mild self-limiting adverse event. No serious adverse events were observed.

Figure 11 - Monash study and private practice patients with knee osteoarthritis exhibit comparable reduced knee pain following intra-articular injection of 50 million (2 x 50 million) versus 100 million (2 xlOO million) autologous MSCs. Line graphs illustrating NPRS scores in patients treated with 2 injections of 50 million autologous hMSCs versus 2 injections of 100 million autologous hMSCs. To date a comparable pain reduction outcome was observed using a dosing protocol of 50 million hMSCs versus 100 million hMSCs. Patient follow-up of those undergoing a 50 Million hMSC protocol has not reached beyond 3 months, but is ongoing. Figure 12 - Monash study and private practice patients with knee osteoarthritis exhibit comparable WOMAC score outcomes following intra-articular injection of 50 million (2 x 50 million) versus 100 million (2 xlOO million) autologous MSCs.

Line graphs illustrating WOMAC outcomes in patients treated with 2 injections of 50 million autologous hMSCs versus 2 injections of 100 million autologous hMSCs. To date a comparable WOMAC outcome was observed using a dosing protocol of 50 million hMSCs versus 100 million hMSCs. Patient follow-up of those undergoing a 50 Million hMSC protocol has not reach beyond 3 months, but is ongoing. Figure 13 - Reduction in knee pain is improved in LaTrobe study and private practice patients treated with microfracture plus intra-articular injection of autologous hMSCs compared to microfracture treatment alone. Line graphs of pain ratings (NPRS) indicate a trend of greater improvement in patients undergoing arthroscopic microfracture plus hMSC therapy versus arthroscopic microfracture alone.

Figure 14 - Functional WOMAC outcomes in knee osteoarthritis are improved in LaTrobe study and private practice patients treated with microfracture plus intra-articular injection of autologous hMSCs compared to microfracture treatment alone. Line graphs of WOMAC outcomes indicate a trend of greater improvement in patients undergoing arthroscopic microfracture plus hMSC therapy versus arthroscopic microfracture alone.

Figure 15 - Magnetic Resonance Imaging (MRI) of patient knee joint one month after abrasion osteoplasty and hMSC injection reveals early immature cartilage formation. MRI scan image shows area of early immature cartilage growth (convex light area indicated by arrow) one month post-abrasion osteoplasty and a single injection of 50 million autologous hMSCs.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all medical, scientific, and technical, terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g. , in medical practice, stem cell biology, anatomy, radiology, surgery, cell culture, cell biology, molecular biology, pharmacology, protein chemistry, and biochemistry). Unless otherwise indicated, the cell culture techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present),

As used herein, the term about, unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, of the designated value.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used in this application, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or". That is, unless specified otherwise, or clear from context, "X employs A or B" is intended to mean any of the natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then "X employs A or B" is satisfied under any of the foregoing instances. Further, at least one of A and B and/or the like generally means A or B or both A and B. In addition, the articles "a" and "an" as used in this application and the appended claims may generally be construed to mean "one or more" unless specified otherwise or clear from context to be directed to a singular form.

The term "adverse event" as used herein, refers to as any undesirable clinical occurrence in a subject/patient (as compared to the subject's baseline health) and is any untoward medical occurrence defined as an unintended disease or injury or untoward clinical signs (including abnormal laboratory findings) in a patient. An adverse event is also any event related to any underlying medical condition, present at baseline, which increases in severity by a clinically meaningful amount after treatment is undertaken. A "mild adverse event" refers to an adverse event that does not interfere with the subject's usual activity or is transient, resolved without treatment, and is without sequelae. A "moderate adverse event" refers to an adverse event that interferes with a subject's usual activity or requires symptomatic treatment. A "severe adverse event" refers to an adverse event causing severe discomfort with significant impact of the subject' s usual activity and requiring treatment.

The term "joint" as used herein, refers to a point of articulation between two or more bones connected by a cartilaginous tissue. Large joints refer to load-bearing joints such as the knee. Medium joints include, but are not limited to, hip, joints, shoulder joints, elbow joints, ankle joints and the Sacro-iliac joint. Small joints includes, but are not limited to, wrist joints, hand joints, foot joints, facet joints and the temporomandibular joint.

The term "human mesenchymal stem cells (hMSCs)" as used herein, refers to a heterogeneous population of multipotent cells found natively in multiple human tissues including adipose, bone marrow, dental pulp, skin, peripheral blood, cord blood and skeletal muscle. Some non-exhaustive characteristics of hMSCs are: (1) differential adherence to tissue culture plastic relative to other cells found in their native tissues; (2) expression of cell surface antigens including CD 105, CD 90 and CD73 and the absence of haematopoetic markers CD34 and CD45; (3) relative immuno-neutrality; (4) proliferation in culture; (5) the ability to differentiate, under suitable culture conditions, into at least chondrocytes, osteoblasts, or adipocytes (see, for example, Caplan et al., 2011). hMSCs can also be cultured and expanded starting from primary hMSCs that are tissue derived. hMSCs generated by expansion in culture are sometimes referred to herein as "cultured-expanded hMSC progeny".

The term "therapeutically effective number" as used herein, refers to a sufficient number of human MSCs being administered which will relieve to some extent one or more of the symptoms of the joint condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of the affected joint. For example, an "effective amount" for therapeutic uses is the number of hMSCs required to provide a clinically significant decrease in disease symptoms, as described herein.

The terms "treating" or "treatment" as used herein, refer to treatment of a human subject by a medical professional (e.g. , by administering a therapeutic agent to the human subject). Also encompassed within the meaning of the term "treating" or "treatment" are prevention or reduction of the disease to be treated, e.g. , by administering a therapeutic at a sufficiently early phase of disease to prevent or slow its progression.

The terms "co-administration" or the like, as used herein, are meant to encompass administration of selected therapeutic agents (e.g. , hMSCs and a growth factor) in treatment regimens in which the agents are administered to the same affected joint by the same route of administration (e.g. , by intra-articular administration) at the same or different times and in combination as a single formulation, or as separate, therapeutic agents.

The term "local administration" as used herein, refers to administration proximal to or within an affected joint, e.g. , by intra-articular administration or intra- discal administration.

Methods of Treatment

The methods described herein include treating a human subject suffering from joint degeneration, by administering hMSCs in a dosing regime and/or in combination with abrasion osteoplasty shown by the inventors to confer therapeutic benefit, while avoiding moderate or severe adverse inflammatory events ("flare ups"), unexpectedly observed at higher doses of hMSCs. While not wishing to be bound by theory, evidence of the capacity of hMSCs to differentiate along a chosen cell lineage pathway represents great promise in the area of regenerative medicine it is postulated that their beneficial effect is also achieved through an immunosuppressive and paracrine mechanism and hence manipulation of the disease process. hMSCs are observed to suppress inflammatory T-cell proliferation and inhibit maturation of monocytes and myeloid dendritic cells resulting in an immunomodulatory and anti-inflammatory effect. They also produce essential cytokines such as TGF-β, Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF) and secrete an array of bioactive molecules that stimulate local tissue repair. Surprisingly, notwithstanding the known immunosuppressive of hMSCs, the inventors found that hMSCs administered in a number expected to be therapeutically effective (100 million) induced a very high frequency of moderate and severe adverse events (e.g. , pain "flare ups"), some of which even required patient hospitalization.

Accordingly, described herein is a method of treating a human subject suffering from joint degeneration, the method comprising local administration to an affected joint of a therapeutically effective number of hMSCs, wherein the number of hMSCs is insufficient to induce a moderate or severe adverse event. In some embodiments of the just-described method the number of hMSCs is insufficient to induce a severe adverse event. In some embodiments the number of hMSCs is insufficient to induce a severe adverse event or a moderate adverse event in greater than 25% of human subjects treated. In other embodiments the number of hMSCs is insufficient to induce a severe adverse event or a moderate adverse event in greater than about 20% to about 5% of human subjects treated, e.g., about 18%, 17%, 15%, 12%, 10%, 8%, or another percentage of human subjects. In some embodiments the hMSC number is insufficient to induce a moderate or serious adverse event in greater than about 10% of human subjects treated.

Also described herein is a method of treating a human subject suffering from joint degeneration, the method comprising performing:

(i) abrasion osteoplasty on an affected joint; and

(ii) local administration of a therapeutically effective number of hMSCs to the affected joint following abrasion osteoplasty.

In some embodiments of any of the above treatment methods the human subject to be treated is suffering from, but not limited to, osteoarthritis, rheumatoid arthritis, a cartilage tear, intervertebral disc inflammation or intervertebral disc degeneration. In particular embodiments the subject to be treated suffers from osteoarthritis. Symptoms, diagnostic tests, and prognostic tests for each of the above-mentioned conditions are known in the art. See, e.g., Harrison's Principles of Internal Medicine ," 19th ed., Vols 1 & 2, 2015, The McGraw-Hill Companies, Inc. Common diagnostic methods for joint conditions include, arthography, magnetic resonance imaging (MRI), blood tests in the case of suspected arthritis for anti citrulline modified proteins (anti- CCP) joint fluid analysis for inflammatory cytokines.

The disease status of a human subject suffering from joint degeneration can be assessed by a number of standardised questionnaires known in the art including, but not limited to, those described below.

A 0-10 numeric pain rating scale (NPRS), which asks participants to rate their knee pain intensity over the previous week. The NPRS has been validated for use in people with knee osteoarthritis (Roos et al., 1998; Ornetti et al., 2011).

The Knee Injury and Osteoarthritis Outcome Score (KOOS) consists of five subscales being pain, other symptoms, function in daily living, function in sport and recreation and knee related quality of life. It is reliable and valid for the population of people with osteoarthritis (Roos et al., 1999).

The Orebro Musculoskeletal Pain (OMP) Questionnaire. This questionnaire has been to shown to be reliable and valid for detecting individuals at risk of developing persistent pain (Linton et al., 2003). It asks questions relating to a variety of known risk factors for the development of chronicity. This questionnaire can be used to assess the potential impact of psychosocial factors on participants' outcome.

A seven-point global perceived effect scale (Beurskens et al., 1996) in which participants are asked to indicate any overall change in their condition from a specified time point (e.g., at the beginning of a clinical trial). Measures of global effect are a recommended outcome measure for clinical trials (Dworkin et al., 2005).

The Western Ontario and McMaster Universities Arthritis Index (WOMAC) is a set of standardised questionnaires to evaluate the condition of patients with osteoarthritis of the knee and hip, including pain, stiffness and physical functioning of the joints. WOMAC measures five items for pain, two items for stiffness and seventeen items for functional limitation. As used herein, the higher the WOMAC score the greater the improvement in joint function.

In some embodiments the treatment method results in regrowth of cartilage at the affected joint within a period of about 12 months from the first local administration of hMSCs, as assessed by, e.g. , MRI or another comparable imaging modality. In other embodiments, within 12 months of a first administration, the treatment results in a reduction of pain in the affected joint of at least about 10% relative to pain experienced at the time of the first administration, e.g. , a reduction in pain of 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or another percent reduction in pain from at least about 10% to about 100% (i.e. , no pain in the affected joint) as assessed by any of the relevant tests described herein (e.g. , NPRS, OMP and WOMAC). In further embodiments, within 12 months of a first administration, the treatment results in an increase in mobility in the affected joint of at least 10% relative to mobility at the time of the first administration, such as an increase in mobility of 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or another percent increase in mobility from at least about 10% to about 100% (i.e. , no stiffness or lack of mobility in the affected joint) as assessed by any of the relevant tests described herein (e.g. , KOOS and WOMAC).

In some embodiments the human subject to be treated is not older than about 60 years of age. In other embodiments the human subject is in an age range from about 40 to about 60 years, e.g. , 42, 45, 48, 50, 53, 55, 56, 57, or another age from about 40 years to about 60 years. In other embodiments the human subject is in an age range of about 18 years to about 50 years, e.g., 20, 22, 25, 30, 32, 35, 37, 40, 42, 45, 48, or another age from about 18 years to about 50 years old.

In some embodiments the affected joint to which hMSCs are administered is a large joint, e.g., a load-bearing joint such as a knee joint. In some embodiments, where the affected joint in the subject to be treated is a large joint, a therapeutically effective number of hMSCs is about 30 million to about 70 million, e.g. , 32 million, 35 million, 40 million, 45 million, 48 million, 50 million, 55 million, 60 million, 65, million, or another number of hMSCs from about 30 million to about 70 million. In other embodiments the number of hMSCs to be administered to an affected large joint is about 40 million to about 60 million, e.g. , 42 million, 45 million, 52 million, 54 million, 57 million, or another number of hMSCs from about 40 million to about 60 million. In some embodiments the number of hMSCs to be administered is about 50 million. In other embodiments is about 40 million.

In some embodiments the affected joint to which hMSCs are administered is a medium or small joint. Examples of medium or small joints that can be treated include, but are not limited to, a hip joint, a shoulder joint, an elbow joint, an ankle joint, a Sacro-iliac joint, a wrist joint, a hand joint, a foot joint, a facet joint, or a temporomandibular joint. In some embodiments, where the affected joint in the subject to be treated is a medium or small joint, a therapeutically effective number of hMSCs is about 5 million to about 20 million, e.g. , 6 million, 7 million, 8 million 9 million, 11 million, 12 million, 13 million, 14 million, 15 million, 16 million, 18 million, 19 million, or another number of hMSCs from about 5 million to about 20 million. In some embodiments the number of hMSCs to be administered is about 7 million to about 15 million, e.g. , 8 million, 9 million, 10 million, 11 million, or another number of hMSCs from about 7 million to about 15 million. In further embodiments the number of hMSCs to be administered is about 9 million to about 12 million, e.g. , 10 million, 11 million, or another number of hMSCs from about 9 million to about 12 million. In some embodiments the number of hMSCs to be administered is about 10 million.

In some embodiments the affected joint in the human subject is an intervertebral disc. In other embodiments, where the affected joint is an intervertebral disc, the number of hMSCs to be administered is about 5 million to about 15 million, e.g. , about 6 million, 7 million, 9 million, 10 million, 11 million, 12 million, 13 million, or another number of hMSCs from about 5 million to about 15 million.

hMSCs are typically administered by intra-articular injection, following local anaesthesia, when administered to a large, medium, or small joint. Generally, such injections are performed with imaging guidance, e.g., by ultrasound imaging, X-ray fluoroscopy, or computerized tomography (CT). Where the affected joint is an intervertebral disc, hMSCs are administered by intra-discal injection, following local anaesthesia. In some embodiments hMSCs are administered in a sterile physiological buffer (e.g. , clinical grade injectable normal saline) in a volume of about 2-4 ml depending on the size of the affected joint to be injected. In other embodiments hMSCs are injected in a sterile carrier solution, e.g. , ACS, as described herein.

In a particular embodiment, the human subject to be treated is suffering from osteoarthritis of the knee and is administered two injections of 100 million autologous hMSCs, wherein the first and second injections are separated by a six month interval. Human Mesenchymal Stem Cells

Suitable hMSCs for use in the treatment methods described herein include those derived from, but nOot limited to, adipose tissue, bone marrow, dental pulp and skin. Progeny hMSCs obtained by culture expansion of primary hMSCs isolated from any of the foregoing tissues are also suitable. Methods for isolation of hMSCs from various tissue sources are known in the art, and generally rely on differential adhesion of hMSCs to tissue culture plastic as compared to other cells native to the same source tissue. See, e.g., Zuk et al. (2001), Francis et al. (2010); Penfornis et al. (2011); Ra et al. (2011) and Alleman et al. (2013).

In an exemplary embodiment hMSCs are isolated from adipose tissue as follows. Lipoaspirate specimens are washed with sterile phosphate saline buffer (PBS) to remove red blood cells, and then subjected to enzymatic digestion using TrypLE Select Enzyme (IX) (ThermoFisher Scientific) at 37 °C with gentle shaking for 1-2 hours to promote tissue disaggregation and then neutralized with mesenchymal stem cell growth media. The digested tissue suspension is then filtered through a sterile nylon mesh (100 μιη) to remove any undigested material. The filtrate is then centrifuged at 1200 x g. The resulting cell pellet is resuspended in mesenchymal stem cell growth media, plated on tissue culture plates and incubated at 37 °C/5% C0₂. Following initial incubation of three days, the tissue culture flasks are washed extensively with sterile DPBS to remove non-adherent cells. The adhered cells are then cultured and expanded in fresh mesenchymal stem cell growth media in a controlled environment until cells are about 80% confluent (about 4- 10 days). The cells are harvested off the tissue culture flasks by digestion with TrypLE Select Enzyme (IX) (ThermoFisher Scientific) at 37 °C for 20 minutes. The purified mesenchymal stem cells are then further expanded into cell factories by re-seeding the cell factories with growth media and incubated at 37 °C in a controlled environment, to obtain higher cell number required for the treatment. Upon reaching 80-90% confluency, the cells are harvested by enzymatic disaggregation of the attached cells using TrypLE Select Enzyme (IX) and incubation at 37 °C for 20 minutes.

Harvested cells are then counted and tested for viability. The isolated cells are also sterility tested to ensure no contamination occurs during laboratory processing.

The cells are characterised by flow cytometry using four surface markers for MSCs (Dominici et al., 2006): CD 90, CD44, CD 73 and CD 105 as positive markers and CD 34 and CD45 as negative surface markers for MSCs to meet international MSCs standard. For cryopreservation, cells are pelleted at 480 x g and washed in PBS 3 times, and then resuspended in cryopreservation medium at 10 million cells/ml, and frozen in Cryo- vials.

In some embodiments the hMSCs to be administered are autologous hMSCs, i.e. , hMSCs derived from the subject to be treated or culture-expanded hMSC progeny thereof. Culture expansion of autologous hMSCs can include about 2 to 20 hMSC culture passages, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 12, 14, 16, 18, or up to 20 passages. In other embodiments, the hMSCs are allogeneic hMSCs or culture-expanded hMSC progeny thereof. Culture expansion of allogeneic hMSCs can include about 2 to 50 hMSC culture passages, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 48 or up to 50 passages. In some embodiments the hMSCs to be administered are hMSCs that were previously cryopreserved, thawed, and washed to remove cryopreservative media shortly before administration (e.g. , within up to 2 hours of administration). In other embodiments the hMSCs to be administered are hMSCs that were harvested directly from hMSC cultures, i.e. , the hMSCs to be administered were not cryopreserved prior to the administration. In some embodiments the cultured hMSCs to be administered are generated directly from parental, primary tissue-derived hMSCs without an intervening cryopreservation step. In other embodiments the cultured hMSCs to be administered were culture-expanded from a thawed, previously cryopreserved hMSC seed stock.

Multiple Administrations

In some embodiments any one of the above-referenced treatment methods includes at least one additional administration of a therapeutically effective number of hMSCs following the first administration, but includes no more than four additional administrations within 18 months of the first administration. In some embodiments the treatment includes two administrations.

In some embodiments the number of hMSCs is the same for each administration. In other embodiments the number of hMSCs is varied between administrations, but falls within the ranges described herein to be therapeutically effective for the joint to be treated (e.g. , a large joint) but insufficient to induce a moderate or severe adverse effect above an indicated frequency.

In some embodiments the interval between a first administration and a second administration is about 4 months to about 8 months, e.g. , 5 months, 6 months, 7 months, or another interval from about 4 months to about 8 months. In some embodiments the interval between a first administration and a second administration is about 6 months. In some embodiments the treatment method includes a total of three administrations of hMSCs within an 18 month period. In some embodiments the treatment method includes a total of 4 administrations of hMSCs within an 18 month period. In other embodiments the treatment method includes a total of 5 administrations of hMSCs within an 18 month period. In some embodiments the interval between all administrations is the same. In some embodiments the treatment includes a first administration of hMSCs and a second administration of hMSCs with an interval of about 6 months in between the administrations. In other embodiments, where hMSCs are administered 3, 4, or 5 times in an 18 month period, at least two different inter-administration intervals are used. In one preferred embodiment 50 million autologous hMSCs are locally administered to the knee joint of a human subject suffering from osteoarthris, wherein the autologous hMSCs are adipose-derived hMSCs or hMSC progeny thereof. In another preferred embodiment 50 million autologous hMSCs are locally administered two times to the knee joint of a human subject suffering from osteoarthris, wherein the two administrations are separated by a six month interval, and wherein the autologous hMSCs are adipose-derived hMSCs or hMSC progeny thereof.

In further embodiments, rather than providing one or more additional hMSC administrations at pre-determined intervals, the subject is given an additional administration of hMSCs based on a determination that joint degeneration is worsening, i.e. , an indication that the subject' s condition is trending towards worse outcomes over a period of time following initial treatment as measured by any of a number of standardized assessments of joint pain and motion, as described herein (e.g. , WOMAC and KOOS) and/or MRI.

Combination Treatments

In some embodiments any of the treatment methods described herein can include local administration of a therapeutic agent in addition to hMSCs. In some embodiments additional therapeutic agents include one or more of a growth factor, cytokine, or glycosaminoglycan. Suitable growth factors include, but are not limited to, at least one of TGF-β Ι, basic fibroblast growth factor (bFGF) and interleukin-1 receptor antagonist (IL-1RA), and any combination of such factors and cytokines. In some embodiments growth factors are provided by administration of autologous conditioned serum (ACS), which have been reported to alleviate osteoarthritis symptoms (Baltzer et al., 2009) and reduce the risk of an immune response as compared to administration of allogeneic preparations of growth factors and cytokines. Suitable glycosaminoglycans include, but are not limited to, hyaluronic acid (HA), heparin sulfate, chondroitin-4- sulfate, chondroitin-6- sulfate, dermatan sulfate, keratin sulfate. In some embodiments the treatment method includes administration of one or more growth factors, but not a glycosaminoglycan. In some embodiments the treatment includes administration of ACS, but not a glycosaminoglycan. In other embodiments the treatment includes administration of both ACS and a glycosaminoglycan (e.g. , HA). In some embodiments the amount of ACS to be used is about 2 ml to 5 ml, e.g., 2.5, 3.3, 3.5, 4.2, 4.5, or another volume of ACS from about 2 ml to about 5 ml.

In some embodiments hMSCs are administered separately from an additional therapeutic agent (e.g. , a growth factor). In some embodiments, where hMSCs and the additional therapeutic agent are administered separately, the additional therapeutic and hMSCs are administered within about 5 minutes to 2 hours of each other, e.g. , 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hours, 1.5 hours, or another time interval of about 5 minutes to two hours. In some embodiments the hMSCs are administered prior to the additional therapeutic agent. In other embodiments the hMSCs are administered following administration of the additional therapeutic agent.

In other embodiments the hMSCs and one or more additional therapeutic agents are co-administered as single combined formulation. In some embodiments hMSCs are combined with ACS prior to administration. Typically, the hMSCs to be administered are resuspended in ACS and administered in a total volume of about 2 ml to 5 ml. In some embodiments hMSCs are combined with both ACS and HA (or another glycosaminoglycan) prior to administration. One of ordinary skill in the art will recognize that such combined formulations are only acceptable when they do not compromise hMSC viability relative to hMSC viability in a formulation that does not include an additional therapeutic agent.

In some embodiments the treatment methods described herein include local administration of hMSCs in subjects that have also received an arthroscopic surgical intervention on the affected joint, which together with hMSCs may enhance the overall efficacy of the treatment method, as compared to administration of hMSCs alone or the arthroscopic surgery alone. In some embodiments hMSCs are administered within about two weeks after arthroscopic surgery, e.g., with the same day, 1 day, 2 days, 3 days, 5 days, 7 days, 8 days, 10 days, 12 days, or within another period within two weeks of the arthroscopic surgery. In some embodiments, where the affected joint is a large, medium, or small joint, but not an intervertebral disc, the arthroscopic surgical intervention at the affected joint is abrasion chondroplasty, abrasion osteoplasty, microfracture, microdrilling, or a combination thereof.

In some embodiments the selected arthroscopic surgical intervention is only abrasion osteoplasty. Abrasion osteoplasty is performed in the affected joint at all sites of full thickness cartilage loss with removal of the subchondral plate/calcified cartilage layer to expose subchondral bone. In some embodiments, after abrasion osteoplasty is performed, microfracture or microdrilling is then performed to the area of exposed subchondral bone. In other embodiments, abrasion chondroplasty is performed to remove calcified cartilage, but which, unlike abrasion osteoplasty, does not significantly perturb the subchondral plate or expose subchondral bone. In some embodiments, after abrasion chondroplasty or osteoplasty, microfracture or microdrilling is then performed on the subchondral bone at the affected joint.

In some embodiments the treatment method avoids the use of microfracture or microdrilling at the affected joint.

EXAMPLES

Example 1 - Evaluation of dosing regimen of autologous human mesenchymal stem cells in the treatment of knee osteoarthritis (Monash University study and private practice patients)

Subjects

In a Monash University- approved clinical study ("Monash Study"), male and female subjects (18-50 years old) suffering from knee osteoarthritis were recruited for a study to determine the treatment efficacy of arthroscopic intra-articular injection to the knee of autologous mesenchymal stem cells (MSCs) at various doses versus no treatment (control). Inclusion criteria for the study included: a radiological diagnosis of osteoarthritis using the American College of Rheumatology criteria; a radiological grading of Grade II-III osteoarthritis of the knee as determined by a qualified radiologist using the Kellgren and Lawrence system; medial compartment osteoarthritis as determined above; osteoarthritis treatment already undertaken defined as: analgesia/anti-inflammatory medication, supplements approved by the treating clinician (e.g. , glucosamine sulphate), an attempted exercise program prescribed by a physiotherapist or medical practitioner for at least eight weeks, weight loss and nutritional management as prescribed by a dietitian or medical practitioner for at least eight weeks, and biomechanical management including bracing if appropriate as prescribed by a physiotherapist, podiatrist or medical practitioner; a minimum pain score of 5 on an 11 -point numerical rating scale; .a maximum Knee Orthopaedic Outcome Score (KOOS) global score of 50; single knee osteoarthritis; less than 5 degrees varus or valgus knee deformity as measured by the long mechanical axis of the knee on X-Ray; and sufficient English skills to complete the questionnaires required for the study, as well as to understand the instructions given by the study doctors.

Exclusion criteria included: pregnancy; breastfeeding; other causes of knee symptoms believed to be due to serious pathology (e.g., a tumour); presence of a blood disorder; ongoing anti-coagulant therapy that could not be ceased safely; MRI- confirmed meniscial tear; MRI-confirmed Grade IV chondral loss; previous meniscectomy/significant partial meniscectomy or other knee related surgery within the previous 12 months; previous intra- articular injectable therapies within the previous six months; lateral compartment and/or patellofemoral compartment Grade 2-4 osteoarthritis; a history of cancer, a history of systemic illness or significant organ impairment/failure (e.g., renal failure); a history of an atypical pain syndrome; a history of infective or inflammatory joint disorders, or suspected infective or inflammatory joint disease; plans at the time of enrolment to undergo surgery in the following 12 months; a history of allergy to any substances used within the treatments; or a history of other musculoskeletal or neurological condition that effects lower limb function.

Consent and baseline assessment

All participants who appeared to be eligible based on the phone screening were invited to attend one of the participating centres for a baseline assessment. Eligible volunteers were provided with the Participant Information Sheet outlining study protocols and procedures. The recruiting doctor answered any questions regarding the study and then asked the volunteer to sign informed consent forms. Baseline assessment included a subjective and physical examination in line with standard medical practice. Participants were then asked to complete an online questionnaire to gain relevant demographic information. Information regarding baseline pain and symptoms was obtained by obtaining a series of outcome questionnaire measures, including the Orebro Musculoskeletal Pain Score, Knees Orthopaedic Outcome Score and Numerical Pain Rating Score using the Clinical Intelligence software (Clinical Intelligence Pty LTD, Caufield South, VIC, Australia)). Patients were also referred for X-Ray and/or MRI scans of their affected knee. Eligible patients were invited to sign a consent form. They were then randomly allocated to one of four groups (Group 1 - Treatment A, Group 2 - Treatment B, Group 3 - Treatment C, and Group 4 - No Treatment Control), as described in detail below. Random allocation was performed using an automated software program and recorded/observed by administration staff not directly involved in the study.

Study structure

Group 1 - Treatment A

A single knee joint intra-articular injection of 100 million autologous mesenchymal stem cells (MSCs) was administered at 0 weeks. Formal function and pain outcome measures were assessed prior to therapy and at 1 month, 3 months, 6 months, and 12 months post commencement of treatment. MRI analysis was performed prior to commencement of therapy and at 6 and 12 months after commencement of therapy.

Group 2 - Treatment B

Two knee joint intra-articular injections of 100 million autologous MSCs were administered at 0 and six months for a total of two hundred million MSCs. Formal function, pain, and MRI outcome measures were assessed as described for Group 1.

Group 3 - Treatment C

Five knee joint intra-articular injections of 40 million autologous MSCs were administered at 0, 1 month, 2 months, 3 months, and 6 months for a total of 200 million MSCs. Measures of pain were assessed prior to and 1 week following each injection. Formal function, pain, and MRI outcome measures were assessed as described for Group 1. Group 4 - Control

Participants in this group received no injectable therapy, but continued conservative current standard of care management of their osteoarthritis. Formal function, pain, and MRI outcome measures were assessed as described for Group 1.

Autologous MSC production method - harvesting and isolation

To harvest adipose derived stromal cells an initial abdominal lipoharvest procedure (liposuction) was performed. Participants received a course of antibiotics commenced the day prior and for 6 days post liposuction. The lateral abdominal region was initially anaesthetised using tumescent fluid comprised of 2% lignocaine with adrenaline, buffered using 8.4% bicarbonate, and suspended in a saline solution. Using a 3 mm lipoaspiration cannula, up to 100 mis of adipose tissue and tumescent fluid were aspirated. The contents of these aspirations were collected in a sterile medical grade single use filter. All patients were reviewed the day after the cell harvest procedure by the treating doctor.

The lipoaspirate, in a sterile single use filter, was transferred to the Magellan Stem Cells (Pty Ltd) laboratory on site at Melbourne Stem Cell Centre. Lipoaspirate specimens were contained within the sterile single use filters and transferred to the laboratory (on site) for further processing. The lipoaspirate were processed in a sterile environment in a Biological Safety Cabinet (BSC) Class II and using strict aseptic technique. All the equipment used was qualified and validated for aseptic use in cell culture and all reagents and buffer used were sterile, qualified and validated for cell culture use.

The lipoaspirate was washed with sterile phosphate saline buffer to remove red blood cells, and then subjected to enzymatic digestion using 0.075% units of collagenase enzyme per ml of lipoaspirate based on the total weight of the lipoaspirate. The digestion was carried out by gentle shaking in an incubator at 37 °C for 1-2 hours. Afterwards, the digested suspension was filtered through sterile nylon (100 μιη) mesh to remove any undigested material. The filtrate was centrifuged twice at 2,000 RPM for 10 minutes, and the cell pellet containing was washed with sterile phosphate buffer, and then resuspended in 10 ml of mesenchymal stem cell medium. The cells were then seeded in a T175 flask and cultured for in a 37 °C/5% C0₂ cell culture incubator until the cells were 80% confluent.

Once the cells were 80% confluent, they were harvested as follows. Cell culture medium was removed from the flask to eliminate non-adherent cells, and the cells were then washed once with sterile Dulbecco's phosphate buffered saline (DPBS). Adherent cells were then removed from the cell culture substrate by incubation with

TrypLE Select Enzyme (IX) (ThermoFisher Scientific) at 37 °C for 15 minutes. Following incubation, the flask was gently tapped to aid in release of the cells, and the enzyme was neutralised with an equal volume of cell culture medium. The resulting cell suspension was then centrifuged at 200 x g for 5 minutes and the pellet was resuspended in cell culture medium. Afterwards, cultures were seeded in pre-warmed complete culture medium at a suitable density. The cells were then cultured for 4-10 days until 80% confluent. The cells were then harvested off of the cell culture flask or cell factory as described above. Harvested cells were counted and tested for viability using a Muse^® Cell Analyzer (Merck-Millipore). In order to ensure the safety and quality of the cultured stem cells, a sample of the cultured cells were sterility tested to ensure no contamination occurred during laboratory processing by inoculating 100 μΐ of sample in to a microbiology culture media for bacteria and fungus growth and sent to a National Association of Testing Authorities (NATA)-accredited testing laboratory.

The cells were characterised by flow cytometry using four surface markers for MSCs as mentioned by the International Society for Cellular Therapy (Dominici et al 2006): CD 90, CD44, CD 73 and CD 105 as positive markers and CD 34 and CD45 as negative surface markers for MSCs.

For cryopreservation, cells were pelleted at 480 x g and resuspended in cryopreservation medium at 10 million cells/ml and frozen in Cryo-vials. Separate dosages containing 100 million autologous MSCs for Groups 1 and 2 and 40 million MSCs for Group 3 were cryopreserved in a liquid nitrogen tank.

Local (intra-articular) administration of MSCs

The initial injection occurred at no less than 10 days post-adipose harvest due to the time required for isolation and expansion of the MSCs. Prior to each intra- articular injection, a single dose of cells was thawed at 37 °C in a sterile water bath and centrifuged to remove cryoprotectant media. The pelleted cells were then mixed with 3 mis of sterile clinical grade injectable normal saline. Upon each visit, the knee was prepared using standard sterile procedures. The area of injection site was first anaesthetised using 2 mis of 1% xylocaine and then MSCs were injected into the knee joint, using a lateral patella-femoral approach under ultrasound guidance to confirm intra-articular placement of the needle.

Participants in Group 1 received autologous MSC injections (100 million cells) at Week 0. Patients in Group 2 received autologous MSC injections (100 million cells per injection) at Week 0 and at 6 months. Patients in Group 3 received autologous MSC injections (40 million cells per injection) at 0, 1, 2, 3 and 6 months.

Autologous blood and synovial fluid sampling

Approximately 34 mis of blood from the antecubital fossa were sampled by the registered medical practitioner/Study nurse three times throughout the study period, at the following visits - baseline, 6 months and 12 months post commencement of the treatment.

Synovial fluid samples, to be used for cytokine and arthritis biomarker analysis,were collected at week 0 (pre-treatment) and at 6 months (post-treatment). The knee will was prepared using standard sterile surgical procedures. The procedural site was infiltrated with 1% xylocaine to improve participant comfort. Using a lateral approach and under ultrasound guidance and standard sterile conditions, a 10ml syringe with 19 gauge needle was directed into the intra- articular space and 2-10 mis of synovial fluid were aspirated and collected.

Follow up, data collection, and data analysis

The Numeral Pain Rating Scale (NPRS) was re-administered one week after each injection. A range of follow up outcomes measures were used for subsequent follow up, including the Knee Orthaedic Outcome Score (KOOS). A central administrator emailed participants regarding completion of online follow-up questionnaires at 1 month, 3 months, 6 months, and 12 months post baseline. If the participant did not have internet access, the questionnaires were posted with a reply paid envelope. Participants who did not return or complete online questionnaires within one week were followed-up by the administrator/research assistant by phone.

Follow up MRIs were performed at 6 and 12 months post commencement of therapy. The following standardised questionnaires were completed by participants in the trial at the designated follow up time points, including pre-treatment/baseline, 1 month, 3 months, 6 months, and 12 months post baseline. The specific outcome measures included as part of follow up are described below.

A 0-10 NPRS, which asks participants to rate their knee pain intensity over the previous week. The NPRS has been validated for use in people with knee osteoarthritis (Roos et al., 1998; Ornetti et al., 2011).

The Orebro Musculoskeletal Pain Questionnaire. This questionnaire has been to shown to be reliable and valid for detecting individuals at risk of developing persistent pain (Linton et al., 2003). It asks questions relating to a variety of known risk factors for the development of chronicity. This questionnaire was used in the current study to assess the potential impact of psychosocial factors on participants' outcome.

Seven-point global perceived effect scale (Beurskens et al., 1996) where participants are asked to indicate any overall change in their condition since the start of the trial. Measures of global effect are a recommended outcome measure for clinical trials (Dworkin et al., 2005). Co-interventions where participants will indicate any other treatments that they have undertaken since the trial began, which will assist in determining whether any differences in outcome may have been influenced by external confounding factors.

Satisfaction with treatment where participants' satisfaction with their treatment, their treatment outcome, and their current status was assessed with three specific questions taken from Deyo's core outcomes questionnaire (Deyo et al., 1998). These questions have been shown to be valid and reliable (Ferrer et al., 2006).

Medication taken in the previous 24-hours. Measuring medication intake of participants is considered a useful outcome measure (Paterson et al., 2005) and it also allows medication intake to be evaluated as a potential confounding factor or co-intervention.

Participants were asked to identify any adverse, harmful, or unpleasant events attributable to the treatment they received in the trial. An open question in the outcome assessment booklet was used to obtain this information from participants (Dworkin et al., 2005).

Patients were identified only by their study number on both questionnaires and their MRI files. Questionnaires were completed by patients on the day of formal review by their treating clinician. The questionnaires were completed alone and on a Clinical Intelligence program, and not during their consultation with the treating clinician.

MRI data was scored by two Radiologists specialised in musculoskeletal imaging. Using 3T MRI T2 cartilage mapping, volumetric 3D gradient and fast spin echo sequences, quantitative data was obtained. A modified Whole-Organ Magnetic Resonance Imaging Score (WORMS) scoring system was used to evaluate the knee with particular attention to the articular cartilage integrity and quality (Peterfy et al., 2004). The MRI and clinical outcome data were entered into a computer spreadsheet by the administrative assistant. Random reliability checks were undertaken by an assigned investigator.

Questionnaires were automatically scored using the Clinical Intelligence software. Data from the questionnaires were stored in a secure offsite database.

The master list that links participants' names with their identification code number was kept securely in a separate location to the questionnaires.

Identifiable personal details (such as participants' names, date of birth, address, contact phone numbers, details of alternative contact people, and the master list linking participant's names with their identification number) were kept securely in three locations. Laboratory cell harvesting, culture, and storage data were recorded for each patient as well. These data included volume of lipoaspirate processed, total number of cells obtained, flow cytometry analysis record, liquid nitrogen inventory list and lot number, supplier and traceability of all the reagents and media used for cell isolation and expansion.

Statistical analyses were conducted using IBM SPSS Statistics 20.0 with a p value of <0.05 considered statistically significant. Descriptive statistics (means and standard deviations for continuous variables and totals with proportions for categorical data) were used to describe the presenting characteristics of included participants. The proportion of participants reporting global improvement (as opposed to no change or worsening) on the global rating of change scale was tested for significance using the one-sample Chi-squared test. To determine whether a significant change in activity limitation was achieved at each of the follow-up times of interest, we used a linear mixed model analysis. This was chosen due to reported strength in analysing longitudinal biological data, including a robust ability to handle missing data (Krueger et al., 2004; Hamer et al., 2009).

Private Patients

The same treatment protocols were also carried out in consented private clinic patients, which, unlike the Monash study, included patients older than 50 years. In some cases private patients, received injections of autologous hMSCs resuspended in autologous conditioned serum (ACS) prepared essentially as described in Baltzer et al. (2008). Results

As shown in Fig. 1, a statistically significant reduction in pain was observed in patients, aged 41-60 years at 1, 3 and 9 months following hMSC therapy (1, 2, or 5 injections). This was observed for both clinical study patients (Fig. 1) and private patients (Fig. 5). Over 65% of patients observed pain improvement greater than 50% and 50% of patients achieved pain relief greater than 75%. Use of qualitative functional measures including WOMAC (Fig. 2) and KOOS (Figs 3 and 4) in study patients and in private patients (Fig. 6, WOMAC) and (Figs. 7 and 8, KOOS) indicated significant improvement in function as early as 1 month following hMSC therapy. Group C subjects, receiving monthly injections of 40 million hMSCs, experienced an unacceptable number of moderate and serious adverse events (data not shown). Thus, the Group C protocol was discontinued. This result suggests that in addition to the number of hMSCs per administration, the interval between administrations is important to minimising moderate and severe adverse events.

Example 2 - Administration of a reduced number of hMSCs to knee osteoarthritis patients greatly reduces adverse effects while maintaining therapeutic efficacy (Monash University study and private practice patients)

Despite the positive therapeutic effects observed in patients receiving injections of 100 million hMSC, 75% of these patients experienced a moderate or severe adverse event including a significant flare up of self-limiting pain and swelling requiring use of strong opiate analgesia. In fact, two patients required hospital admission for pain relief. The distribution of adverse side effects (mild, moderate and severe) in patients (clinical study and private patients) receiving 100 million hMSC injections is illustrated in Fig. 9.

In view of the high incidence of moderate and severe adverse effects observed in patients receiving 100 million hMSC injections, the inventors determined whether a reduced number of hMSCs (50 million) would reduce adverse effects while maintaining therapeutic efficacy. As shown in Fig. 10, the reduced number of hMSCs reduced the incidence of moderate adverse events dramatically reduced to 8%, with no severe adverse events observed at all. Importantly, the efficacy of the reduced dose treatment was unchanged with respect to both pain reduction (Fig. 11) and functional improvement (Fig. 12).

Example 3 - hMSC therapy in conjunction with knee microfracture surgery appears to have has greater efficacy than microfracture surgery alone (LaTrobe University study and private practice patients)

Microfracture has become a commonly practiced surgical technique to assist a healing response in damaged joints. This technique involves making multiple holes (microfractures) into the subchondral plate at the site of a full thickness chondral defect. This exposes bone marrow derived pluripotent cells to the articular surface and creates an environment amenable to healing. Multiple studies have successfully shown a cartilaginous response at the sites of microfracture, yet histology has confirmed that this tissue is fibrocartilage rather than the typical hyaline cartilage of articular surfaces. Whilst evidence suggests effective short-term functional improvement of knee function, long term results are inconclusive with inadequate defect fill and cartilage repair tissue quality (fibrocartilage versus hyaline cartilage) being postulated as reasons for poor long. Thus, the inventors sought to determine whether microfracture treatment in conjunction with intra-articular injection of hMSCs would lead to improved outcomes relative to patients receiving microfracture treatment alone.

In a LaTrobe University-approved clinical study ("LaTrobe Study"), male and female subjects (18-50 years old) having full thickness cartilage defects in a knee were recruited for an unblinded study comparing pain and function in people who have undergone traditional arthroscopic knee surgery (microfracture) alone for a cartilage lesion versus people who underwent the same surgery followed by intra-articular injection of autologous mesenchymal stem cells (MSCs) in their knee. It was also determined whether stem cell injection improved cartilage growth relative to treatment by arthroscopic surgery alone.

After receiving written informed consent from all study participants, each subject was randomly allocated to one of the two groups (surgery alone or surgery plus hMSCs). Volunteers returned to their orthopaedic surgeon to have their planned microfracture surgery. Initially, volunteers in the hMSC treatment arms received four injections occurring at monthly intervals (40 million autologous hMSC per injection) and again at 6 months post arthroscopy for a total of 200 million hMSCs. However, it was noted early in the study, that subjects receiving monthly injections showed a high incidence of moderate or serious adverse events. Thus, the protocol was switched to a two injection (0 and 6 month) protocol in which 50 million autologous hMSCs were administered per injection Activity and pain outcome measures were assessed in the hMSC plus microfracture treated groups versus the control group (microfracture only) at similar time points over the study period. The same protocols were also carried out in consented, private practice patients.

As shown in Fig. 13 (pain assessment) and Fig. 14 (functional assessment), a trend has been observed to date for greater improvement in pain and function in those receiving MSC therapy in addition to microfracture surgery, as compared to that observed in patients receiving microfracture surgery alone.

Example 4 - hMSC therapy in coni unction with knee abrasion osteoplasty induces early immature cartilage formation

In ongoing work, private patients underwent arthroscopic of the area of arthritis with abrasion osteoplasty using an arthroscopy burr to expose the subchondral bone. Within two weeks of the procedure patients received an intra-articular injection of 50 million autologous hMSCs. Patients will receive a second injection of 50 million autologous hMSCs within six months of the initial injection. In preliminary patient results, an example of which is shown Fig. 15, early cartilage formation was observed by MRI at the treated joint (indicated grey zone) suggesting that abrasion osteoplasty in combination with hMSC injection may facilitate cartilage regrowth. Subjects will be administered a second injection of hMSCs within six months of the initial injection and will be imaged at regular intervals, throughout the study.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from AU 2016900743 filed 29 February 2016, the entire contents of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Claims

1. A method of treating a human subject suffering from joint degeneration, the method comprising local administration to an affected joint of a therapeutically effective number of human mesenchymal stem cells, wherein the number of human mesenchymal stem cells is insufficient to induce a moderate or severe adverse event.

2. A method of treating a human subject suffering from joint degeneration, the method comprising performing:

(i) abrasion osteoplasty on an affected joint; and

(ii) local administration of a therapeutically effective number of human mesenchymal stem cells to the affected joint following abrasion osteoplasty.

3. The method of claim 1 or claim 2, wherein the human subject is suffering from osteoarthritis, rheumatoid arthritis, a cartilage tear, intervertebral disc inflammation or intervertebral disc degeneration.

4. The method of claim 3, wherein the affected joint is affected by osteoarthritis.

5. The method of any one of claims 1 to 4, wherein the affected joint is an intervertebral disc.

6. The method of any one of claims 1 to 4, wherein the affected joint is a large joint.

7. The method of claim 6, wherein the large joint is a knee joint.

8. The method of claim 6 or claim 7, wherein the therapeutically effective number of human mesenchymal stem cells administered to the large joint is about 30 million to about 70 million.

9. The method of claim 8, wherein the therapeutically effective number of human mesenchymal stem cells administered to the large joint is about 40 million to about 60 million.

10. The method of any one of claims 1 to 4, wherein the affected joint is a medium joint or small joint.

11. The method of claim 10, wherein the medium joint or small joint is a hip joint, a shoulder joint, an elbow joint, an ankle joint, a Sacro-iliac joint, a wrist joint, a hand joint, a foot joint, a facet joint, or a temporomandibular joint.

12. The method of claim 10 or claim 11, wherein the therapeutically effective number of human mesenchymal stem cells administered to the medium or small joint is about 5 million to about 20 million.

13. The method of claim 12, wherein the therapeutically effective number of human mesenchymal stem cells administered to the medium or small joint is about 7 million to about 15 million.

14. The method of claim 13, wherein the therapeutically effective number of human mesenchymal stem cells administered to the medium or small joint is about 9 million to about 12 million.

15. The method of any one of claims 1 to 14, wherein the human mesenchymal cells are adipose tissue-derived human mesenchymal stem cells or culture-expanded human mesenchymal stem cell progeny thereof.

16. The method of any one of claims 1 to 15, further comprising at least one local additional administration of a therapeutically effective number of human mesenchymal stem cells following the first administration, but no more than four additional local administrations within 18 months of the first administration.

17. The method of claim 16, wherein the interval between the first local administration and a second local administration is about 4 months to about 8 months.

18. The method of claim 17, wherein the interval between the first local administration and the second local administration is about 6 months.

19. The method of claim 16 comprising the at least one additional local administration, wherein the additional local administration is provided following a determination that the joint degeneration is worsening.

20. The method of any one of claims 1 to 19, further comprising local administration to the joint of a growth factor, cytokine or glycosaminoglycan.

21. The method of claim 20, wherein the growth factor or cytokine is selected from the group consisting of: TGF-βΙ, basic fibroblast growth factor (bFGF), interleukin-1 receptor antagonist (IL-1RA) and any combination thereof.

22. The method of claim 20 or claim 21, wherein local administration of the growth factor comprises local administration of autologous conditioned serum.

23. The method of any one of claims 20 to 23, wherein the glycosaminoglycan is hyaluronic acid (HA).

24. The method of any one of claims 1 or 3 to 23, wherein the affected joint underwent abrasion osteoplasty prior to the local administration.

25. The method of any one of claims 1 or 3 to 23, further comprising performing abrasion osteoplasty on the affected joint prior to the local administration.

26. The method of any one of claims 1 to 25, wherein the method does not comprise microfracture or microdrilling on the affected joint.

27. The method of any one of claims 1 to 25, further comprising performing microfracture or microdrilling on the affected joint.

28. The method of any one of claims 1 to 27, wherein the human mesenchymal stem cells are autologous human mesenchymal stem cells.

29. The method of any one of claims 1 to 27, wherein the human mesenchymal stem cells are allogeneic human mesenchymal stem cells.

30. The method of any one of claims 1 to 29, wherein the human mesenchymal stem cells were not cryopreserved prior to the local administration.

31. The method of any one of claims 1 to 30, wherein the treatment results in regrowth of cartilage at the affected joint within a period of 12 months from the first administration.

32. The method of any one of claims 1 to 31, wherein within 12 months of a first administration, the treatment results in a reduction of pain in the affected joint of at least about 10% relative to the pain experienced at the time of the first administration.

33. The method of any one of claims 1 to 32, wherein the treatment results in an increase in mobility in the affected joint of at least 10% within 12 months of the first administration.

34. The method of any one of claims 1 to 33, wherein the human subject is not older than about 60 years.

35. The method of claim 34, wherein the human subject is about 40 to about 60 years old.

36. Human mesenchymal stem cells when used for the treatment of joint degeneration in a human subject according to the method of any one of claims 1 to 35.

37. Human mesenchymal stem cells in a dose therapeutically effective for the treatment of joint degeneration in a human by local administration to an affected joint, wherein the dose comprises an insufficient number of human mesenchymal stem cells to induce a moderate or severe adverse event.

38. Use of human mesenchymal stem cells for the manufacture of a medicament for the treatment of joint degeneration in a human, the medicament comprising a therapeutically effective number of human mesenchymal stem cells for local administration to an affected joint, wherein the therapeutically effective number of human mesenchymal stem cells is insufficient to induce a moderate or severe adverse event.

39. Human mesenchymal stem cells in a dose therapeutically effective for the treatment of joint degeneration in a human subject by administration of the hMSCs to an affected joint following abrasion osteoplasty.

40. Use of human mesenchymal stem cells for the manufacture of a medicament for the treatment of joint degeneration, wherein the medicament is administered to an affected joint following abrasion osteoplasty.